We will use deoxythymidine kinase-deficient (dTK-) mouse 3T3 cells biochmically transformed to the dTK+ phenotype (3T3BT) by equine herpes virus type 1 (EHV-1) by means of selecting mutant clones of HSV-1 that are resistant to the viral dTK-dependent nucleosides arabinosylthymine (araT), acycloguanosine (ACG), and 5-ethyldeoxyuridine (EdU) by virtue of alterations in the HSV-1-specific DNA polymerase (DAN pol). In 3T3BT cells, these are phosphorylated by the EHV-1 dTK respective triphosphates independent of the expression of the HSV-1 dTK. Because of this, HSV-1 that had lost its dTK would still not escape inhibition of replication by the nucleoside, and the dTK- state would afford it little if any selective advantage. In contrast, HSV-1 that had an altered DNA pol that was not inhibited by a given nucleoside triphosphate would be selected for in this system. By this means we plan to obtain a collection of clones of HSV-1 with altered DNA pol, and to determine the patterns of cross-resistance with araT, ACG, EdU, as well as bromovinyldeoxyuridine (BVdU), arabinosyladenine (araA), phosphonoacetate (PAA), and phosphonoformate (PFA). The data will help provide the bases for selecting the best pairs of drugs for combination therapy of HSV-1 in order to minimize the appearance of drug-resistant mutants.